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Fluorescent Sensor Detects Drug-Induced Protein Stress in Cells

BioPhotonicsDec 2017
STATE COLLEGE, Pa. — A fluorescent sensor that will become activated in a cell when misfolded proteins in the cell begin to aggregate could be an effective way to detect drug-induced proteome stress prior to cell death.

Researchers designed an unstable protein, called AgHalo, to sense proteome stress through the protein’s aggregation. AgHalo was tagged with a fluorogenic ligand that fluoresced when AgHalo formed soluble aggregates. When the AgHalo protein began to misfold and aggregate, the dye interacted with the hydrophobic portions of the protein and began to fluoresce.

Image of cells expressing the AgHalo sensor before (left) and after (right) cellular stress. The AgHalo sensor is turned on when misfolded proteins begin to aggregate and provides a quantitative measure of cellular stress that can be used to evaluate drug safety. Courtesy of Yu Liu, Penn State University.The researchers used their sensor to test the level of protein stress caused by five commonly-used anti-cancer drugs. All five produced some level of protein stress detectable by the AgHalo sensor before any cell death was observed.

“Because we tested the anti-cancer drugs at much higher doses than typically used for treatment, our results do not necessarily call into question the continued use of these drugs,” said researcher Yu Liu. “However, because protein stress from long-term treatments could have lasting effects, evaluating drugs with our new sensor will help in the development of safer drugs.”

Drug safety is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. According to the researchers, previous systems have used sensors that were always “on.” The cells would have a diffuse fluorescence prior to any stress, and the system could only detect protein stress when misfolded proteins aggregated, forming bright spots of fluorescence that were large enough to be seen under a microscope.

“An additional advantage of our system is that the level of fluorescence is correlated to the amount of protein aggregation in the cell, so we can quantify the level of stress,” said Liu. “Also, because our method measures the level of fluorescence, rather than having to identify the fluorescence under a microscope, it can be done using more accessible technology, like plate readers, and it is much more high-throughput.”

Professor Xin Zhang said, “Drug-induced protein stress in cells is a key factor in determining drug safety. Drugs can cause proteins — which are long strings of amino acids that need to be precisely folded to function properly — to misfold and clump together into aggregates that can eventually kill the cell. We set out to develop a system that can detect these aggregates at very early stages and that also uses technology that is affordable and accessible to many laboratories.”

“With our method, we can quantitatively detect protein stress in cells at much earlier stages and therefore researchers can begin to study the mechanisms that cells use to combat this stress and develop compounds that can enhance the cell’s ability to handle protein stress,” said Zhang.

The research was performed at Penn State University. The research was published in Angewandte Chemie International Edition (doi: 10.1002/ange.201702417).

The emission of light or other electromagnetic radiation of longer wavelengths by a substance as a result of the absorption of some other radiation of shorter wavelengths, provided the emission continues only as long as the stimulus producing it is maintained. In other words, fluorescence is the luminescence that persists for less than about 10-8 s after excitation.